Modification of low density lipoprotein (LDL) particles and their interaction with the molecules of the extracellular matrix and foam cell formation are key processes in the development of the atherosclerotic lesions. Our studies address LDL modifications in atherosclerosis as well as extracellular lipoprotein particles in human atherosclerotic arteries and aortic stenosis. Stenotic aortic valves provide an interesting focus for our studies, as such valves are characterized by atherosclerosis-like lesions, consisting of activated inflammatory cells including T lymphocytes, macrophages, and mast cells, and of lipid deposits, calcific nodules, and bone tissue. We also study structural modifications of high density lipoproteins (HDL) induced by proteases present in the arterial intima. Such proteases affect the function of HDL in stimulating cholesterol efflux from macrophage foam cells, and so may impair removal of cholesterol from the intima.
We propose that by promoting cholesterol accumulation and plaque vulnerability and by locally regulating hemostasis, mast cells in atherosclerotic lesions have the potential to contribute to the clinical outcomes of atherosclerosis, such as myocardial infarction and stroke.
We have identified several novel factors which are capable of triggering inflammatory reaction in arterial wall. Cholesterol crystals are a common constituent of atherosclerotic lesions already early in the process of atherogenesis. We have studied their possible significance in the pathogenesis of atherosclerosis. One of the important aims of the project is to identify suitable targets for the novel anti-inflammatory therapies to treat atherosclerosis.
LDL in atherogenesis
Modification of low density lipoprotein (LDL) particles and their interaction with the molecules of the extracellular matrix and foam cell formation are key processes in the development of the atherosclerotic lesions. In our studies, we are examining the effects of various lipases (phospholipase A2 and sphingomyelinase) and proteases (mast cell proteases and cathepsins) on LDL particles. These modifications lead to aggregation and fusion of LDL, enhance the binding of LDL to human aortic proteoglycans and induce foam cell formation. Since the extracellular pH in atherosclerotic lesions can decrease locally and so generate microdomains having an acidic pH, we have also examined the effect of pH on these processes. Most recently, we have particularly focused on the proinflammatory roles of modified LDL and acidic pH. Based on our present results, it appears that all these key processes of atherosclerosis are significantly enhanced at acidic pH.
In our current projects, we examine the mechanisms of LDL aggregation with the aim of inhibiting LDL modification and aggregation with the aim of inhibiting atherogenesis. We also apply lipidomics to study the individual differences in LDL modification and aggregation, particularly in LDL samples obtained from persons who consume either animal fat or vegetable oils. We are also isolating extracellular lipoprotein particles from human atherosclerotic arteries and stenotic aortic valves to be able to identify the lipoprotein modifications that occur during the development of atherosclerosis and aortic stenosis.
Aortic stenosis: from molecular mechanisms to a pharmacological trial
Stenotic aortic valves are characterized by atherosclerosis-like lesions consisting of activated inflammatory cells, which include T lymphocytes, macrophages, and mast cells. In addition, the lesions contain lipid deposits, calcific nodules, and even bone tissue. Active mediators of calcification and cells with osteoblast-like activity are present in diseased valves. Extracellular matrix remodeling, including collagen synthesis and elastin degradation by matrix metalloproteinases and cathepsins, contributes to leaflet thickening and stiffening. In experimental animals, hypercholesterolemia induces calcification and bone formation in aortic valves, which can be inhibited by statin treatment. The potential of statins to retard progression of aortic valve stenosis has also been recognized in clinical studies; however, the results of such trials have been negative. We discovered that angiotensin II-forming enzymes are upregulated in stenotic valves. Thus, angiotensin II may participate in profibrotic progression of aortic valve stenosis and may serve as a possible therapeutic target of the disease. Based on the above original observation in our laboratory, we launched together with the Division of Cardiology at the University of Helsinki (Principal Investigator Professor Markku Kupari), a blinded, randomized, placebo-controlled, prospective study, the aim of which is to determine the influence of effective treatment with Type 1 angiotensin II (Ang II) receptor (AT-1R) antagonist, using candesartan (target dose 16 mg) on stenotic aortic valves. The investigators will specifically quantify whether candesartan attenuates the key pathogenic mechanisms of aortic valve stenosis, namely inflammation, fibrosis, elastin degradation, calcification, and neovascularization.
HDL in atherogenesis
A major culprit in atheroma development is an accelerated cholesterol-laden macrophage (foam cell) formation. By promoting cellular cholesterol release (efflux), high density lipoproteins (HDL) initiate the reverse cholesterol transport (RCT) pathway from macrophage foam cells located in the arterial intima to liver for biliary excretion. Both quantity and quality of the circulating HDL particles matter for their optimal antiatherogenic potential. We study how proteolysis of various HDL subpopulations can disrupt the anti-atherogenic functions of HDL.
Mast cells are a potent source of serine proteases in the human intima. We have demonstrated that the mast cell-derived chymase is able to proteolytically modify HDL particles in vitro leading to the loss in their function as macrophage cholesterol acceptors. The small lipid-poor preβ-migrating HDL subpopulation is the most susceptible to proteolysis. Since preβ-HDL efficiently stimulate cholesterol efflux via the ABCA1 receptor pathway, proteolytic depletion of preβ impaired HDL functionality. Recently we found that acute systemic stimulation of mast cells in the mouse (anaphylactic shock) reduced the abilities of serum and peritoneal fluid to promote cholesterol efflux from cultured macrophage foam cells. Moreover, by applying a macrophage-specific RCT assay, we have expanded our in vivo models to evaluate the impact of HDL proteolysis on the entire RCT pathway. Our experimental in vivo systems include genetically-modified mice such as the mast-cell deficient W-sash c-kit mutant mice, and pathophysiological models that may affect RCT such as the physical restraint stress in mice. More recently, acute psychological stress was found to inhibit cholesterol absorption in mice. This effect also enhanced the macrophage-specific RCT in the stressed mice. At present, we focus on experimental conditions capable to stimulate in vivo the release of cholesterol from lipid-loaded cells involved in atherogenesis. This approach includes cholesterol efflux studies using different cell type cultures and treatment of mice with protease-resistant cholesterol acceptors or with agents that improve HDL flux in interstitial fluids.
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Innate immunity in atherosclerosis
Chronic inflammation is recognized as a major driving force in atherogenesis, yet the factors that trigger and sustain the inflammatory reaction in arterial wall still remain elusive. The aim of the research group is to identify factors that initiate and perpetuate the inflammation in the arterial wall, and on the other hand, factors potentially inhibiting the inflammatory process.
During atherogenesis, cholesterol accumulation in the arterial wall causes formation of cholesterol crystals already at early stages of lesion development. We have shown that cholesterol crystals are strong activators of inflammasome signaling cascade in human macrophages, resulting in secretion of the key proinflammatory cytokine interleukin-1β (Rajamäki et al. 2010). Thus we demonstrated for the first time that cholesterol crystals are not just inert by-products of the disease process but possess strong proinflammatory potential. In chronic inflammatory diseases, levels of the acute phase protein serum amyloid A (SAA) are increased up to 1000-fold, and elevated levels of SAA are associated with the risk of atherosclerosis. We have shown that SAA is capable of activating inflammasome-mediated interleukin-1β secretion in human macrophages (Niemi et al. 2011). Thus SAA may exert a direct local effect on atherogenesis via proinflammatory activation of macrophages. Furthermore, we have shown that local extracellular acidosis in atherosclerotic lesions activates the inflammasome pathway and synergizes with both SAA and cholesterol crystals in induction of interleukin-1β secretion (Rajamäki et al. 2013).
We have also identified mechanisms that potentially restrict inflammation in the arterial wall. We have shown that ethanol inhibits the responses of innate immunity by inducing mast cell apoptosis (Nurmi et al. 2009), and we are currently focusing on the effects of ethanol on macrophage-mediated chronic inflammation.
The treatment of atherosclerosis is focused on decreasing the levels of blood lipids and currently there are no drugs which would specifically target the inflammation in atherosclerosis. Thus our goal is to identify suitable targets for the novel anti-inflammatory therapies to treat atherosclerosis.
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Mast cells in atherogenesis
Mast cells, better known as allergy cells, are proinflammatory effector cells also present in the human arterial intima and in evolving atherosclerotic lesions. Our understanding of the relationship between the proatherogenic activities of arterial mast cells (MCs) and the development of atherosclerotic lesions is advancing. Experiments in vitro and in vivo experiments in animals, and immunohistologic studies of human coronary samples have uncovered mechanisms by which activated mast cells could participate in the development of the lesions. When activated, mast cells acutely expel a fraction of their cytoplasmic granules, which are filled with a wide selection of heparin-bound preformed mediators, notably histamine and neutral proteases. Activation of mast cells also induced synthesis and secretion of newly formed mediators including lipid mediators (eicosanoids) and cytokines. The microenvironmental targets of these effector molecules are various lipoprotein particles in the intimal fluid, components of the extracellular matrix, and intimal cells neighboring the activated mast cells. We propose that by promoting cholesterol accumulation and plaque vulnerability and by locally regulating hemostasis, MCs in atherosclerotic lesions have the potential to contribute to the clinical outcomes of atherosclerosis, such as myocardial infarction and stroke.
We have developed an efficient method to grow human mast cells from progenitor cells isolated from human peripheral blood. Such cultured mast cells are invaluable tools for exploring the mechanisms by which mast cells affect their microenvironment. Currently, the neutral proteases and lipid mediators released by activated mast cells in culture are in the focus, when aiming to understand the role of mast cells in atherogenesis.